PROJECT SUMMARY Noncoding RNAs (ncRNAs) have revolutionized biomedical research as master regulators of all known biological processes through the modulation of target gene expression. Furthermore, ncRNAs are attractive candidates for the treatment of human diseases. However, current ncRNA research and development is limited to the use of chemically-engineered or -synthesized ncRNA agents (e.g., miRNAs and siRNAs) that are decorated with various and extensive artificial modifications. This is in accordance with the termination of a number of siRNA/miRNA oligonucleotide drugs from further clinical investigations, due to the induction of adverse immune responses in patients. Although the exact causes of immunogenicity remain undisclosed, artificial modifications have been well documented to provoke immunogenicity as recognized by toll like receptors. Therefore, we have made large efforts to develop novel bioengineering techniques for the production of natural ncRNA agents. Our efforts have led to the identification of stable tRNA/pre-miRNA hybrid carriers for large-scale expression of recombinant miRNAs and siRNAs in a common strain of E. coli. Our studies have demonstrated that bioengineered miRNA/siRNA agents (BERAs) carry no or just a few natural posttranscriptionally-modified nucleosides and exhibit favorable stability within human cells. Furthermore, BERAs are biologically active in modulating target gene expression and cancer cellular processes (e.g., cell proliferation and chemosensitivity, etc.) in vitro, and suppress tumor progression in animal models in vivo. Given these exciting preliminary findings, we hypothesize that the tRNA/pre-miRNA-based technology may be refined and standardized to achieve a more consistent, higher-yield production of fully-humanized natural RNAi agents. To test this hypothesis, we will optimize pre-miRNA to improve expression yield (>30% of total RNAs) and incorporate human tRNAs to offer fully-humanized ncRNA carriers (Aim 1). In addition, we will develop a standardized pipeline to produce a collection (20-30) of ready-to-use BERAs, and critically compare the efficacy and safety of model BERAs with synthetic miRNA mimics (Aim 2). This project will establish a novel ncRNA bioengineering platform toward high-yield production of fully-humanized RNAi agents and offer BERAs as a new class of natural ncRNA agents for research and development.